Microcapsules comprising liver cells and erythropoietin, methods for preparing said microcapsules and methods for treating a patient comprising applying said microcapsules to the patient

ABSTRACT

Microcapsules containing a capsule shell encapsulating a suspension of a therapeutically effective amount of liver cells in physical contact to a liver cell stimulating amount of erythropoietin.

TECHNICAL FIELD

The invention relates to microcapsules comprising liver cells anderythropoietin, methods for preparing said microcapsules and methods fortreating a patient comprising applying said microcapsules to thepatient.

BACKGROUND OF THE INVENTION

Although the liver of a healthy subject is able to regenerate itself byrepairing or replacing injured or ill tissue, unfortunately once acertain amount of liver cells has died or has been severely damagedthrough disease or injuries, the whole organ may fail. Such a failure,be it an acute or chronic failure, may cause disease and death. Thetherapy of liver diseases encompasses conventional means, such as theadministration of drugs. However, it is also the state of the art totransplant livers or parts thereof. Furthermore, it is widely known totransplant liver cell populations in patients, such as described forinstance in WO 2004/009766 A2. It is, however, still a major challengein devising therapies for curing acute or chronic hepatic diseases todeliver liver cells to a patient in need thereof, which methods areoptimized in terms of viability, engraftment, proliferation anddifferentiation of the transplanted liver cells in the target tissue.

Haque et al. (Biotechnology Letters 27 (5) (2005), 317-322)) describe invitro studies of alginate-chitosan microcapsules as an alternative toliver cell transplants for the treatment of liver failure.Chandrasekaran et al. (Tissue Engineering 12 (7), (2006)) disclosehepatic progenitor cells embedded in electrostatically produced beads.

In spite of the efforts to develop therapeutic systems which efficientlydeliver liver cells into the patient's target tissue, there stillremains the need to provide more reliable therapeutic means to cureliver diseases, in particular means which provide a high viability andphysiological activity of the liver cells in the target body.

SUMMARY OF THE INVENTION

The present teaching solves said problem by providing microcapsulescomprising a capsule shell, preferably a biocompatible capsule shell,encapsulating a suspension of a therapeutically effective amount ofliver cells in physical contact to a liver cell stimulating amount oferythropoietin. In a preferred embodiment the present invention providesmicrocapsules comprising a capsule shell and a core, wherein the capsuleshell encapsulates in the core a suspension of a therapeuticallyeffective amount of liver cells and a liver cell stimulating amount oferythropoietin, in particular wherein the erythropoietin (in thefollowing termed EPO) and the liver cells are in physical contact toeach other so as to provide a stimulating effect of the EPO to the livercells.

Thus, in a preferred embodiment the present invention foresees toprovide microcapsules comprising a capsule shell, preferably made from abiocompatible capsule shell material, and a core being enveloped by saidcapsule shell. In one preferred embodiment of the present invention thecore contains a suspension of a therapeutically effective amount ofliver cells in physical contact to a liver cell stimulating amount oferythropoietin.

In another preferred embodiment the core contains a matrix, preferablymade from a biocompatible matrix material, wherein the suspension of thetherapeutically effective amount of liver cells is embedded in saidmatrix in physical contact to a liver cell stimulating amount oferythropoietin. Preferably, the material of the matrix is the same asthe capsule shell material. In another embodiment the biocompatiblematrix material may be another material than the capsule shell material.

Thus, the present invention provides the teaching to encapsulate botherythropoietin and liver cells together in a biocompatible capsule shellsuch that the erythropoietin is in physical contact to the liver cellsso as to exert at least one of its biological functions on the livercells, in particular in stimulating the liver cells.

One advantage of the present invention is that the liver cells areencapsulated in the capsule shell and therefore do not elicit anyadverse reaction, in particular allergic or immunological reaction, inthe patient, in which the microcapsule is preferably transplanted.Furthermore, the close contact of the erythropoietin to the liver cellstimulates said liver cells to perform their biological function so asto provide the patient with the biological functions of a liver.

In a particularly preferred embodiment the liver cells are contained inthe microcapsule in such a concentration so as to be in physical contactto each other providing an even more pronounced effect when stimulatedby the erythropoietin. Thus, the invention foresees in a particularlypreferred embodiment that the liver cells contained in the microcapsulesare in physical contact to each other and are in physical contact to theerythropoietin.

DETAILED DESCRIPTION

In the context of the present invention the expression “liver cells arein physical contact to erythropoietin” means that erythropoietin is ableto exert at least one of its biological functions on the liver cell, inparticular is able to reach and being reversibly or irreversibly boundby the receptors for erythropoietin present on the liver cell.

In the context of the present invention the expression “liver cellsbeing in physical contact to each other” means that the liver cells arepresent in the microcapsule of the present invention in such a closevicinity to each other that the cells touch each other and provide astable environment closely resembling the natural physiologicalsituation in a liver.

In the context of the present invention the term “stimulating the livercells” means that the erythropoietin increases the biologicalfunctionality of the liver cells, preferably in the patient in which themicrocapsule is transplanted, increases the viability of the livercells, increases their storage stability and/or increases theirpotential to successfully perform their biological function once beingtransplanted in the subject.

In the context of the present invention “biocompatibility” means thatthe material, in particular the capsule shell material and/or the matrixmaterial is able to keep the integrated liver cells viable and allow theinteraction between the erythropoietin and the liver cells. In aparticularly preferred embodiment the term “biocompatible” means thatthe material allows, preferably a long-term, implantation in a patientwhile still retaining the function of the embedded liver cells withouteliciting any undesirable local or systemic effect in the subject, inparticular allergic and immunological reactions. In a particularlypreferred embodiment the term “biocompatible” means that the material isable to perform as a substrate supporting the liver cell activityincluding the solicitation of molecular and mechanical similar systembetween the liver cells and the EPO, preferably in order to optimizeliver regeneration without eliciting any undesirable effects in thecells and the subject.

In the context of the present invention the term “erythropoietin”designates a glycoprotein hormone that controls erythropoieses or bloodcell production, preferably a substance that, in appropriately dosage,controls the growth, differentiation and maturation of stem cells viaerythroblasts to erythrocytes.

Erythropoietin is a glycoprotein having 166 amino acids, threeglycosylation sites and a molecular weight of about 34,000 Da. DuringEPO-induced differentiation of erythrocyte progenitor cells, globinsynthesis is induced, synthesis of the heme complex is augmented and thenumber of ferritin receptors is increased. Thereby the cell can take upmore iron and synthesize functional hemoglobin. In mature erythrocytes,hemoglobin binds oxygen. Thus the erythrocytes and the hemoglobincontained therein play a key role in supplying oxygen to the organism.These processes are initiated through the interaction of EPO with anappropriate receptor on the cell surface of the erythrocyte progenitorcells (Graber and Krantz, Ann. Rev. Med. 29 (1978), 51-56).

In the context of the present invention the term “erythropoietin” bothencompasses the wild type erythropoietin, in particular the humanerythropoietin, and derivatives therefrom. In the context of the presentinvention derivatives of erythropoietin are recombinant erythropoietinproteins which are characterized by at least one amino acid deviation,in particular deletion, addition or substitution of one more amino acidcompared to the wild type EPO, and/or erythropoietin proteins with adifferent glycosylation pattern compared to the wild typeerythropoietin. In a particularly preferred embodiment erythropoietinderivatives are also fusion proteins or truncated proteins of wild typeerythropoietin or derivatives thereof. In a particularly preferredembodiment a derivative of erythropoietin is also a wild typeerythropoietin having a different glycosylation pattern compared to thewild type glycosylation pattern.

The term “erythropoietin” used here includes EPO of every origin,especially human or animal EPO. The term used here thus encompasses notonly the naturally occurring, or in other words wild-type forms of EPO,but also its derivatives, also termed modifications, muteins or mutants,as long as they exhibit the biological effects of wild-typeerythropoietin.

In connection with the present invention, there will be understood by“derivatives” also those derivatives of erythropoietin that, whileretaining the basic erythropoietin structure, are obtained bysubstitution of one or more atoms or molecular groups or residues,especially by substitution of sugar chains such as ethylene glycol,and/or whose amino acid sequences differ from that of the naturallyoccurring human or animal erythropoietin protein in at least oneposition but essentially have a high degree of homology at the aminoacid level and comparable biological activity. Erythropoietinderivatives which can be employed, for example, in the present inventionare known from WO 94/25055, EP 0148605 B1 or WO 95/05465.

“Homology” means especially a sequence identity of at least 80%,preferably at least 85% and particularly preferably at least more than90%, 95%, 97% and 99%. The term “homology” known by the person skilledin the art thus refers to the degree of relationship between two or morepolypeptide molecules. This is determined by the agreement between thesequences. Such agreement can mean either identical agreement or else aconservative exchange of amino acids.

According to the invention, the term “derivative” also includes fusionproteins, in which functional domains of another protein are present onthe N-terminal part or on the C-terminal part. In one embodiment of theinvention, this other protein may be, for example, GM-CSF, VEGF, PIGF, astatin or another factor that has a stimulating effect on endothelialprogenitor cells. In a further embodiment of the invention, the otherprotein may also be a factor that has a stimulating effect on livercells.

The differences between an erythropoietin derivative and native or wildtype erythropoietin may arise, for example, through mutations such asdeletions, substitutions, insertions, additions, base exchanges and/orrecombinations of the nucleotide sequences coding for the erythropoietinamino acid sequences. Obviously such differences can also be naturallyoccurring sequence variations, such as sequences from another organismor sequences that have mutated naturally, or mutations introducedselectively into the nucleic acid sequences coding for erythropoietin,using common means known in the art, such as chemical agents and/orphysical agents. In connection with the invention, therefore, the term“derivative” also includes mutated erythropoietin molecules, or in otherwords erythropoietin muteins.

According to the invention, peptide or protein analogs of erythropoietinmay also be used. In connection with the present invention, the term“analogs” includes compounds that do not have any amino acid sequenceidentical to the erythropoietin amino acid sequence but have athree-dimensional structure greatly resembling that of erythropoietin,so that they have comparable biological activity. Erythropoietin analogsmay be, for example, compounds that contain, in a suitable conformation,the amino acid residues responsible for binding of erythropoietin to itsreceptors, and that are therefore able to simulate the essential surfaceproperties of the erythropoietin binding region. Compounds of this typeare described, for example, in Wrighton et al., Science, 273 (1996),458.

The EPO used according to the invention can be produced in various ways,for example by isolation from human urine or from the urine or plasma(including serum) of patients suffering from aplastic anemia (Miyake etal., J.B.C. 252 (1977), 5558). As an example, human EPO can also beobtained from tissue cultures of human renal cancer cells (JA UnexaminedApplication 55790/1979), from human lymphoblast cells, which have theability to produce human EPO (JA Unexamined Application 40411/1982), andfrom a hybridoma culture obtained by cell fusion of a human cell line.EPO can also be produced by methods of gene technology, using suitableDNA or RNA coding for the appropriate amino acid sequence of EPO toproduce the desired protein by genetic engineering, for example in abacterium, in a yeast, or in a plant, animal or human cell line. Suchmethods are described, for example, in EP 0148605 B2 or EP 0205564 B2and EP 0411678 B1.

In the context of the present invention derivatives of erythropoietinare in a preferred embodiment functional and clinically proven EPOderivatives, preferably selected from the group consisting of Epoetin,also termed Procrit, Epogen, Eprex or NeoRecormon, Epoetin delta, alsotermed Dynepo, Darbepoetin, also called Aranesp, PDpoetin, CERA(continuous erythropoietin receptor antagonist) and methoxy polyethyleneglycol-epoetin beta, also termed Mircera.

In a preferred embodiment of the present invention a microcapsule is a,preferably spherical, bead made from a biocompatible capsule shellmaterial which contains embedded therein the therapeutically effectiveamount of liver cells in physical contact to a liver cell stimulatingamount of erythropoietin. In the context of the present invention amicrocapsule is preferably a sphere, preferably with a diameter from 140μm to 10 mm, preferably 50 μm to 1 mm, preferably 60 μm to 800 μm,preferably 100 to 700 μm. In a preferred embodiment of the presentinvention the microcapsules of the present invention consist of theliver cells, the erythropoietin and the biocompatible capsule shellmaterial. Preferably in addition to the above three elements a coatingof the microcapsule and/or a biocompatible matrix in the core of themicrocapsule is foreseen.

Thus, in a particularly preferred embodiment the present inventionrelates to microcapsules comprising a therapeutically effective amountof liver cells encapsulated in a biocompatible matrix material inphysical contact to a liver cell stimulating amount of erythropoietin.

In a particularly preferred embodiment, the liver cells are embedded inthe capsule shell in form of a suspension, preferably in a cell culturesuspension. The liver cell suspension is preferably in form of livercells contained in a cell culture medium or in a physiologicallyacceptable aqueous solution. The liver cell suspension is preferably asuspension of liver cells in a cell culture medium.

In a particularly preferred embodiment, the liver cells are embedded inthe matrix contained in the shell in form of a suspension, preferably ina cell culture suspension, preferably in form of liver cells containedin a cell culture medium or in a physiologically acceptable aqueoussolution.

In a particularly preferred embodiment the present invention relates tomicrocapsules, wherein the liver cells are selected from the groupconsisting of hepatic precursor cells, hepatic stem cells, hepatoblastsand hepatocytes.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the liver cells are obtained from adult liver,embryogenic liver, fetal liver, neonatal liver or liver cell cultures.Preferably, the liver cells are living liver cells. In a preferredembodiment, the liver cells may be obtained from a living or a dead, inparticular a recently deceased, donor.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the liver cells are human liver cells, non-humanprimate liver cells, pig liver cells, dog liver cells, cat liver cells,rabbit liver cells, mouse liver cells or rat liver cells.

In a particularly preferred embodiment the present invention relates tomicrocapsules, wherein the microcapsules have an average diameter from100 to 700 μm, preferably from 200, 300, 400, 500, 600 or 650 to 700 μm.

In a particularly preferred embodiment the present invention relates tomicrocapsules, wherein the therapeutically effective amount of livercells is 10⁴ to 10⁸, preferably 10⁵ to 10⁷ liver cells/ml suspension.

In a particularly preferred embodiment the present invention relates tomicrocapsules, wherein the liver cells have an average diameter of 8 to14 μm, preferably 9 to 12 μm.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the liver cell stimulating amount oferythropoietin is 10⁻⁷ to 10⁻³, preferably 10⁻⁷ to 10⁻⁵ and preferably10⁻⁶ to 10⁻⁵ U/ml suspension.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the EPO is wild type erythropoietin orrecombinant erythropoietin.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the capsule shell material is selected from thegroup consisting of alginate, alginate-chitosan (AC),alginate-poly-L-lysine (APA), thermogelation-polymer and PEG-hydrogel.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein the matrix material is selected from the groupconsisting of alginate, alginate-chitosan (AC), alginate-poly-L-lysine(APA), thermogelation-polymer and PEG-hydrogel.

In a furthermore preferred embodiment the present invention relates tomicrocapsules, wherein in addition to the liver cells at least onefurther cell type is present in the microcapsule.

In a particularly preferred embodiment the at least one further celltype present in the microcapsule is selected from the group consistingof liver stellate cells, biliary cells, hemopoietic cells, monocytes,macrophage lineage cells, lymphocytes and endothelial cells.

In a furthermore preferred embodiment of the present invention it isforeseen that the microcapsule comprises in addition to the liver cellsand the EPO at least one further growth factor, preferably selected fromthe group consisting of HGH (human growth factor), VEGF (vascularendothelial growth factor), CSF (colony stimulating factor),thrombopoietin, SCF complex (Skpcollien, F-box containing complex), SDF(stromal cell-derived factor-1), NGF (nerve growth factor), PIGF(phosphatidyl inositol glycan anchor biosynthesis, class F), HMGcoreductase inhibitor, ACE (angiotensin converting enzyme) inhibitor,AT-1-inhibitor and an NO donor.

In a particularly preferred embodiment the present invention relates tomicrocapsules, wherein the microcapsules are coated.

In a particularly preferred embodiment of the present invention, thecoating is a polymer coating, a sugar coating, a sugar alcohol coatingand/or a fat or wax coating.

In a furthermore preferred embodiment the present invention relates to amethod for preparing the microcapsules according to the abovecomprising:

-   -   a) providing a suspension of a therapeutically effect amount of        liver cells and a liver cell stimulating amount of        erythropoietin,    -   b) mixing the suspension of the liver cells and the        erythropoietin to bring them in physical contact to each other        and    -   c) encapsulating the suspension of the liver cells and the        erythropoietin in a capsule shell material so as to form the        microcapsule.

In a furthermore preferred embodiment the present invention relates to amethod, wherein the microcapsules obtained in step c) are cryopreserved.

In a furthermore preferred embodiment the present invention relates to amethod for the prophylactic or therapeutic treatment of a liver diseasein a subject in need thereof comprising administering the microcapsulesaccording to the present invention to the subject in need thereof.

In a particularly preferred embodiment the present invention relates toa method, wherein the liver disease is hepatitis, cirrhosis, inbornerrors of metabolism, acute liver failure, acute liver infections, acutechemical toxicity, chronic liver failure, cholangiocitis, biliarycirrhosis, Alagille syndrome, alpha-1-antitrypsin deficiency, autoimmunehepatitis, biliary atresia, cancer of the liver, cystic disease of theliver, fatty liver, galactosemia, gallstones, Gilbert's syndrome,hemochromatosis, hepatitis A, hepatitis B, hepatitis C and otherhepatitis viral infections, poryphyria, primary sclerosing cholangitis,Reye's syndrome, sarcoidosis, tyrosinemia, type 1 glycogen storagedisease or Wilson's disease.

In a furthermore preferred embodiment the present invention relates to amethod, wherein the administration is effected by introduction of themicrocapsules under the liver capsule, into the spleen, into the liver,into the liver pulp or into the spleenic artery or portal vein.

In a furthermore preferred embodiment the present invention relates to amethod for introducing liver cells into a subject comprisingadministering the microcapsules according to the present invention intothe subject.

In a particularly preferred embodiment of the present invention theadministration is effected by introduction into the subject in needthereof, wherein the administration is a topical, enteral or parenteraladministration. In a preferred embodiment the topical administration isan epicutaneous, inhalational, vaginal or internasal administration. Ina preferred embodiment the enteral administration is by mouth, bygastric feeding tube or rectally. In a preferred embodiment theparenteral administration is by injection or infusion, preferablyintervenous, intraarterial, intramuscular, intracardiac, subcutaneous,intraosseous, intradermal, intrathecal, intraperitoneal or intravesicaladministration. In a furthermore preferred embodiment the parenteraladministration is transdermal, transmucosal or inhalational.

In a furthermore preferred embodiment the present invention relates to amethod for cultivating liver cells in a culture medium, preferably invitro, comprising culturing microcapsules according to the presentinvention in a suitable culture medium and under conditions suitable formaintaining or increasing viability of the liver cells.

Further preferred embodiments of the present invention are the subjectmatter of subclaims.

1. A microcapsule comprising a capsule shell encapsulating a suspensionof a therapeutically effective amount of liver cells in physical contactto a liver cell stimulating amount of erythropoietin.
 2. Themicrocapsule according to claim 1, wherein the liver cells are selectedfrom the group consisting of hepatic precursor cells, hepatic stemcells, hepatoblasts and hepatocytes.
 3. The microcapsule according toclaim 1, wherein the liver cells are obtained from adult liver,embryogenic liver, fetal liver, neonatal liver or liver cell cultures.4. The microcapsule according to claim 1, wherein the liver cells arehuman liver cells, non-human primate liver cells, pig liver cells, dogliver cells, cat liver cells, rabbit liver cells, mouse liver cells orrat liver cells.
 5. The microcapsule according to claim 1, wherein themicrocapsules have an average diameter from 100 to 700 μm.
 6. Themicrocapsule according to claim 1, wherein the liver cells have anaverage diameter of 8 to 14 μm.
 7. The microcapsule according to claim1, wherein the therapeutically effective amount of liver cells is from10⁴ to 10⁸ liver cells/ml suspension.
 8. The microcapsule according toclaim 1, wherein the liver cell stimulating amount of erythropoietin is10⁻⁷ to 10⁻³ U/ml suspension.
 9. The microcapsule according to claim 1,wherein the erythropoietin is wild type erythropoietin or recombinanterythropoietin.
 10. The microcapsule according to claim 1, wherein thecapsule shell is made from a biocompatible material being selected fromthe group consisting of alginate, alginate-chitosan (AC),alginate-poly-L-lysine (APA), thermogelation-polymer and PEG-hydrogel.11. The microcapsule according to claim 1, wherein in addition to theliver cells at least one further cell type is present in themicrocapsule.
 12. The microcapsule according to claim 1, wherein themicrocapsule is coated.
 13. The microcapsule according to claim 1,wherein the microcapsule contains a biocompatible matrix embedding theliver cells and the erythropoietin.
 14. A method for preparing themicrocapsule according to claim 1, said method comprising d) providing asuspension of a therapeutically effect amount of liver cells and a livercell stimulating amount of erythropoietin, e) mixing the suspension ofthe liver cells and the erythropoietin to bring them in physical contactto each other and f) encapsulating the suspension of the liver cells andthe erythropoietin in a biocompatible capsule shell material so as toform the microcapsule.
 15. The method according to claim 14, wherein themicrocapsules obtained in step c) are cryopreserved.
 16. A method forthe prophylactic or therapeutic treatment of a liver disease in asubject in need thereof comprising administering the microcapsulesaccording to claim 1 to the subject in need thereof.
 17. The methodaccording to claim 16, wherein the liver disease is hepatitis,cirrhosis, inborn errors of metabolism, acute liver failure, acute liverinfections, acute chemical toxicity, chronic liver failure,cholangiocitis, biliary cirrhosis, Alagille syndrome,alpha-1-antitrypsin deficiency, autoimmune hepatitis, biliary atresia,cancer of the liver, cystic disease of the liver, fatty liver,galactosemia, gallstones, Gilbert's syndrome, hemochromatosis, hepatitisA, hepatitis B, hepatitis C and other hepatitis viral infections,poryphyria, primary sclerosing cholangitis, Reye's syndrome,sarcoidosis, tyrosinemia, type 1 glycogen storage disease or Wilson'sdisease.
 18. The method according to claim 15, wherein theadministration is effected by introduction of the microcapsules underthe liver capsule, into the spleen, into the liver, into the liver pulpor into the spleenic artery or portal vein.
 19. A method for introducingliver cells in a subject comprising administering the microcapsulesaccording to claim 1 into the subject.
 20. A method for cultivatingliver cells in a culture medium comprising culturing microcapsulesaccording to claim 1 in a suitable culture medium.